PROJECT SUMMARY/ABSTRACT The objective of this proposal is to understand the interaction of ?-crystallin with membrane cholesterol (Chol) and cholesterol bilayer domains (CBDs) in the fiber-cell plasma membranes of the human eye lens. CBDs are formed in the fiber-cell plasma membrane of the eye lens and have positive physiological functions, helping to maintain lens transparency and possibly protect against cataract formation. The soluble lens protein, ?-crystallin, is a major structural protein that, under healthy conditions, forms a transparent lattice in the lens and plays a major role in maintaining lens transparency. Several discoveries report that the level of ?-crystallin in the lens cytoplasm declines with age and cataract progression, accompanied by a corresponding increase in the amount of membrane-bound ?-crystallin. However, the mechanism by which ?-crystallin associates with fiber-cell plasma membrane and how the age-related change in membrane lipid composition affects the ?-crystallin binding is unclear. I hypothesize that the binding of ?-crystallin to membrane is inhibited by CBDs, which decreases the light scattering and helps maintain lens transparency. In their proposed role, CBDs should increase the level of ?-crystallin in the lens cytoplasm favoring its chaperone function and maintaining lens cytoplasm homeostasis. I discovered that the properties of CBDs change significantly with age and are related to the size of the CBD, which increases with age and is greater in nuclear than in cortical membranes. Based on my extensive experience working with CBDs in model and human lens membranes, I will (i) determine the lipid composition in fiber-cell plasma membranes that promotes or inhibits the binding of ?-crystallin to membranes, (ii) test the hypothesis that CBDs inhibit the binding of ?-crystallin to membranes, and finally (iii) determine the effects of CBD on the binding of ?-crystallin in clear and cataractous human lens membranes of different age groups. The analysis will include donor's health history, sex, and race. I developed electron paramagnetic resonance (EPR) methods to study small-volume aqueous biological samples (3 L at X-band or 150 nL at W-band), particularly for studies of lens membranes obtained from the eyes of a single donor. This technique provides a major advantage when studying the binding of ?-crystallin in membranes of age-matched clear and cataractous lenses from human donors. In addition, the EPR approach has the unique ability to simultaneously provide information about the CBDs and the binding of ?-crystallin. For the last eight years, my research has focused on understanding the molecular organization of lipids and proteins in plasma membrane of intact fiber cells of human eye lenses. Building upon the knowledge I acquired during these studies, here I propose moving my research in a new direction to focus on the interaction of CBDs with ?-crystallin. There is a clear need for a more in-depth understanding of the roles of CBDs in the binding of ?-crystallin in the fiber cell plasma membrane. The findings from this study will help fill this gap and produce valuable insights in maintaining lens transparency.